1. Field of the Invention
The present invention relates generally to β-sheet mimetics, including inhibitors of tryptase in treating inflammation and several other disorders as well as to a chemical library of β-sheet mimetics.
2. Description of the Related Art
The β-sheet conformation (also referred to as a β-strand conformation) is a secondary structure present in many polypeptides. The β-sheet conformation is nearly fully extended, with axial distances between adjacent amino acids of approximately 3.5 Å. The β-sheet is stabilized by hydrogen bonds between NH and CO groups in different polypeptides sheets. Additionally, the dipoles of the peptide bonds alternate along the sheets, which imparts intrinsic stability to the β-sheet. The adjacent sheets in the β-sheet can run in the same direction (i.e., a parallel β-sheet) or in opposite directions (i.e., an antiparallel β-sheet). Although the two forms differ slightly in dihedral angles, both are sterically favorable. The extended conformation of the β-sheet conformation results in the amino acid side chains protruding on alternating faces of the β-sheet.
The importance of β-sheets in peptides and proteins is well established (e.g., Richardson, Nature 268:495–499, 1977; Halverson et al., J. Am. Chem Soc. 113:6701–6704, 1991; Zhang, J. Biol. Chem. 266:15591–15596, 1991; Madden et al., Nature 353:321–325, 1991). The β-sheet is important in a number of biological protein-protein recognition events, including interactions between proteases and their substrates.
Inhibitors that mimic the β-sheet structure of biologically active proteins or peptides would have utility in the treatment of a wide variety of conditions. For example, trypsin-like serine proteases form a large and highly selective family of enzymes involved in hemostasis/coagulation (Davie, E. W. and K. Fujikawa, “Basic mechanisms in blood coagulation,” Ann. Rev. 799–829, 1975) and complement activation (Muller-Eberhard, H. J., “Complement,” Ann. Rev. Biochem. 44:697–724, 1975). Sequencing of these proteases has shown the presence of a homologous trypsin-like core with amino acid insertions that modify specificity and which are generally responsible for interactions with other macromolecular components (Magnusson et al., “Proteolysis and Physiological Regulation,” Miami Winter Symposia 11:203–239, 1976).
Tryptase, a trypsin-like serine protease found exclusively in mast cells, has attracted much interest due to its potential role as a mediator of inflammation. For example, in the lung, tryptase is released along with other mediators of inflammation in response to binding of an inhaled antigen to cell-surface IgE receptors (Ishizaka and Ishizaka, Prog. Allergy 34:188–235, 1984). Tryptase has also been shown to cleave vasoactive intestinal peptide in vitro (Caughey et al., J. Pharmacol. Exp. Ther. 244:133–137, 1988; Tam and Caughey, Am. J. Respir. Cell Mol. Biol. 3:27–32, 1990). These results suggest that tryptase may increase bronchoconstriction via proteolysis of bronchodilating peptides in asthma patients. Consistent with this hypothesis is the recent finding that synthetic tryptase inhibitors blocked airway responses in allergic sheep (Clark et al., Am. J. Respir. Crit. Care Med. 152:2076–2083, 1995).
Tryptase activates extracellular matrix-degrading proteins prostromelysin (pro-MMP-3) and procollagenase (pro-MMP-1) via MMP-3, suggesting a role for the enzyme in tissue remodeling and inflammation (Gruber et al., J. Clin. Invest. 84:8154–8158, 1989) and, therefore, possibly in rheumatoid arthritis. Additionally, prostromelysin, when activated, has been shown to degrade the extracellular matrix around atherosclerotic plaques. Since abnormally high levels of tryptase-containing mast cells have been found in coronary atheromas, tryptase may play a role in atheromatous rupture (release of the thrombus), the final event of coronary atherosclerosis (Kaartinen et al., Circulation 90:1669–1678, 1994).
Other activities of tryptase include the following. Tryptase cleaves fibrinogen but is not inactivated in the presence of endogenous proteinase inhibitors (Schwartz et al., J. Immunol. 135:2762–2767, 1985; Ren et al., J. Immunol. 159:3540–3548, 1997), and may function as a local anticoagulant. It has been demonstrated to be a potent mitogen for fibroblasts and may be involved in pulmonary fibrosis and interstitial lung disease (Ruoss et al., J. Clin. Invest. 88:493–499, 1991). Tryptase may also be responsible for the activation of PAR-2 (proteinase activated receptor-2) on endothelial cells and keratinocytes (Molino et al., J. Biol. Chem. 272:4043–4049, 1997).
Inhibition of intestinal motility, especially colonic motility, is a major complication of abdominal surgery. The condition, termed post-operative ileus, delays the normal resumption of food intake after surgery and often leads to prolonged hospitalization. Mast cells are pro-inflammatory cells that are normally present in the wall of the intestine. Manipulation of intestine and intestinal inflammation are accompanied by influx and degranulation of mast cells in the wall of the intestine. Mast cell tryptase and chymase are proteases that account for 25% of the total protein of mast cells (Schwartz et al., J. Immunol. 138:2611–2615, 1987). They are released from mast cell upon degranulation within the wall of the colon. A method of treating or preventing post-operative ileus was discovered based on the observation that PAR-2 is expressed in colonic muscle cells, and that activation of PAR-2 inhibits colonic motility. Since the PAR-2 receptor is activated, at least in part, by tryptase, inhibition of tryptase could be an effective method of treating post-operative ileus (U.S. Pat. Nos. 5,958,407 and 5,888,529).
Given the central role of mast cells in allergic and inflammatory responses, inhibition of tryptase may result in significant therapeutic effects. Inhibitors of tryptase may be useful for preventing or treating asthma, pulmonary fibrosis and interstitial pneumonia, nephritis, hepatic fibrosis, hepatitis, hepatic cirrhosis, scleroderma, psoriasis, atopic dermatitis, chronic rheumatoid arthritis, influenza, Crohn's disease, ulcerative colitis, inflammatory bowel disease, nasal allergy, and atherosclerosis.
While significant advances have been made in the synthesis and identification of conformationally constrained, β-sheet mimetics (U.S. Pat. Nos. 6,245,764, 6,117,896 and 6,020,331 and published PCT WO00/11005 and WO99/41276), there is still a need in the, art for small molecules that mimic the secondary structure of peptides. There is also a need in the art for libraries containing such members, particularly those small templates capable of supporting a high diversity of substituents. In addition, there is a need in the art for techniques for synthesizing these libraries and screening the library members against biological targets to identify bioactive library members. Further, there is a need in the art for small, orally available inhibitors of tryptase, for use in treating inflammatory diseases, central nervous system disorders, as well as several other disorders. In particular, there is a need for inhibitors of tryptase for use in the treatment or prevention of various mammalian disease states, for example asthma, cough, chronic obstructive pulmonary disease (COPD), bronchospasm, emesis, neurodegenerative disease, ocular disease, inflammatory diseases such as arthritis, central nervous system conditions such as anxiety, migraine and epilepsy, nociception, psychosis, and various gastrointestinal disorders such as Crohn's disease.
The present invention fulfills these needs and provides further related advantages.